Glycerol free ethanol production

ABSTRACT

The invention relates to a recombinant cell, preferably a yeast cell comprising: a) one or more heterologous genes encoding a glycerol dehydrogenase activity; b) one or more genes encoding a dihydroxyacetone kinase (E.C. 2.7.1.28 and/or E.C. 2.7.1.29); c) one or more heterologous genes encoding a ribulose-1,5-biphosphate carboxylase oxygenase (EC 4.1.1.39, RuBisCO); and d) one or more heterologous genes encoding a phosphoribulokinase (EC 2.7.1.19, PRK); and optionally e) one or more heterologous genes encoding for a glycerol transporter. This cell can be used for the production of ethanol and advantageously produces little or no glycerol.

FIELD

The invention relates to a recombinant cell suitable for ethanolproduction, the use of this cell for the preparation of ethanol and/orsuccinic acid, and a process for preparing fermentation product usingsaid recombinant cell.

BACKGROUND

Microbial fermentation processes are applied for industrial productionof a broad and rapidly expanding range of chemical compounds fromrenewable carbohydrate feedstocks. Especially in anaerobic fermentationprocesses, redox balancing of the cofactor couple NADH/NAD⁺ can causeimportant constraints on product yields. This challenge is exemplifiedby the formation of glycerol as major by-product in the industrialproduction of—for instance—fuel ethanol by Saccharomyces cerevisiae, adirect consequence of the need to reoxidize NADH formed in biosyntheticreactions. Ethanol production by Saccharomyces cerevisiae is currently,by volume, the single largest fermentation process in industrialbiotechnology, but various other compounds, including other alcohols,carboxylic acids, isoprenoids, amino acids etc., are currently producedin industrial biotechnological processes. For conventional fermentativeproduction of fuel ethanol, such as from corn starch and cane sugar,sugars predominantly occur as dimers or polymers of hexose sugars, whichupon release in monosaccharides after pretreatment and enzymatichydrolysis by different forms of glucohydrolases can be efficiently andrapidly fermented by Saccharomyces cerevisiae. Cellulosic or secondgeneration bioethanol is produced from e.g. lignocellulosic fractions ofplant biomass that is hydrolyzed intro free monomeric sugars, such ashexoses and pentoses, for fermentation into ethanol. Apart from thesugar release during pretreatment and hydrolysis of the biomass, sometoxic by-products are formed depending on several pretreatmentparameters, such as temperature, pressure and pre-treatment time.Various approaches have been proposed to improve the fermentativeproperties of organisms used in industrial biotechnology by geneticmodification. A major challenge relating to the stoichiometry ofyeast-based production of ethanol, but also of other compounds, is thatsubstantial amounts of NADH-dependent side-products (in particularglycerol) are generally formed as a by-product, especially underanaerobic and oxygen-limited conditions or under conditions whererespiration is otherwise constrained or absent. It has been estimatedthat, in typical industrial ethanol processes, up to about 4 wt % of thesugar feedstock is converted into glycerol (Nissen et al. Yeast 16(2000) 463-474). Under conditions that are ideal for anaerobic growth,the conversion into glycerol may even be higher, up to about 10%.

Glycerol production under anaerobic conditions is primarily linked toredox metabolism. During anaerobic growth of S. cerevisiae, sugardissimilation occurs via alcoholic fermentation. In this process, theNADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenasereaction is re-oxidized by converting acetaldehyde, formed bydecarboxylation of pyruvate to ethanol via NAD⁺ dependent alcoholdehydrogenase. The fixed stoichiometry of this redox-neutraldissimilatory pathway causes problems when a net reduction of NAD⁺ toNADH occurs elsewhere in metabolism (e.g. biomass formation). Underanaerobic conditions, NADH re-oxidation in S. cerevisiae is strictlydependent on reduction of sugar to glycerol. Glycerol formation isinitiated by reduction of the glycolytic intermediate dihydroxyacetonephosphate (DHAP) to glycerol 3-phosphate (glycerol-3P), a reactioncatalyzed by NAD⁺ dependent glycerol 3-phosphate dehydrogenase.Subsequently, the glycerol 3-phosphate formed in this reaction ishydrolysed by glycerol-3-phosphatase to yield glycerol and inorganicphosphate. Consequently, glycerol is a major by-product during anaerobicproduction of ethanol by S. cerevisiae, which is undesired as it reducesoverall conversion of sugar to ethanol. Further, the presence ofglycerol in effluents of ethanol production plants may impose costs forwaste-water treatment.

WO2013/89878 describes a recombinant cell functionally heterologousnucleic acid sequences encoding for ribulose-1,5-phosphatecarboxylase/oxygenase (EC 4.1.1.39; herein abbreviated as “RuBisCO”),and optionally molecular chaperones for RuBisCO, and phosphoribulokinase(EC 2.7.1.19; herein abbreviated as “PRK”).

WO2015/107496 describes a recombinant cell functionally expressingheterologous nucleic acid sequences encoding for RuBisCO-units RbcL,RbcS and RcbX, molecular chaperones for Rubisco GroEL and GroES. In theexamples PRK is expressed with a tetracyclin-inducible promoter TetO7.

DESCRIPTION OF THE FIGURES

FIG. 1: Glycerol re-uptake pathway integrated at genomic locus INT1 withCRISPR-Cas9. The figure depicts the integration at the genomic site INT1aided by the CRISPR-Cas9 methodology described in PCT/EP2016/050136.INT1-5′: 500 bp 5′-integration flank for INT1 locus PCR-amplified fromCEN.PK113-7D; Sc_DAK1: expression cassette Sc_DAK1 PCR-amplified frompDB1333; Ec_gldA: expression cassette E. coli gldA PCR-amplified frompDB1332; Dr_T3: D. rerio T3 glycerol transporter expression cassette;Zr_T5: Z. rouxii T5 glycerol transporter expression cassettePCR-amplified from pDB1336; INT1_3′: 500 bp down stream integrationflank for INT1-locus; (a)(b)(c)(d): 50 bp connector sequences flankingthe different expression cassettes to enable correct assembly of thepathway at INT1; GT INT1: genomic target sequence for the Cas9 induceddouble strand break.

FIG. 2: Fermentation profiles of strains IME324, IMX774, DS78742,DS78743, DS787444 on a mineral medium supplemented with approximately 50g glucose per liter; initial pH of medium was 4.6. Levels of residualglucose (g/L; solid squares, black line) and formed biomass (g/L; opendiamonds, grey line), glycerol (g/L; open triangles, black line), aceticacid (g/L; open squares, grey line) and ethanol (g/L; open circles,black line) were measured every 4 hours during a 32 h fermentation run.

DETAILED DESCRIPTION

The term “a” or “an” as used herein is defined as “at least one” unlessspecified otherwise.

When referring to a noun (e.g. a compound, an additive, etc.) in thesingular, the plural is meant to be included. Thus, when referring to aspecific moiety, e.g. “gene”, this means “at least one” of that gene,e.g. “at least one gene”, unless specified otherwise. The term ‘or’ asused herein is to be understood as ‘and/or’.

When referring to a compound of which several isomers exist (e.g. a Dand an L enantiomer), the compound in principle includes allenantiomers, diastereomers and cis/trans isomers of that compound thatmay be used in the particular method of the invention; in particularwhen referring to such as compound, it includes the natural isomer(s).

The term ‘fermentation’, ‘fermentative’ and the like is used herein in aclassical sense, i.e. to indicate that a process is or has been carriedout under anaerobic conditions. Anaerobic conditions are herein definedas conditions without any oxygen or in which essentially no oxygen isconsumed by the cell, in particular a yeast cell, and usuallycorresponds to an oxygen consumption of less than 5 mmol/l·h⁻¹, inparticular to an oxygen consumption of less than 2.5 mmol/l·h⁻¹, or lessthan 1 mmol/l·h⁻¹. More preferably 0 mmol/L/h is consumed (i.e. oxygenconsumption is not detectable. This usually corresponds to a dissolvedoxygen concentration in the culture broth of less than 5% of airsaturation, in particular to a dissolved oxygen concentration of lessthan 1% of air saturation, or less than 0.2% of air saturation.

The term “cell” refers to a eukaryotic or prokaryotic organism,preferably occurring as a single cell. The cell may be selected from thegroup of fungi, yeasts, euglenoids, archaea and bacteria.

The cell may in particular be selected from the group of generaconsisting of yeast.

The term “yeast” or “yeast cell” refers to a phylogenetically diversegroup of single-celled fungi, most of which are in the division ofAscomycota and Basidiomycota. The budding yeasts (“true yeasts”) areclassified in the order Saccharomycetales, with Saccharomyces cerevisiaeas the most well-known species.

The term “recombinant (cell)” or “recombinant micro-organism” as usedherein, refers to a strain (cell) containing nucleic acid which is theresult of one or more genetic modifications using recombinant DNAtechnique(s) and/or another mutagenic technique(s). In particular arecombinant cell may comprise nucleic acid not present in acorresponding wild-type cell, which nucleic acid has been introducedinto that strain (cell) using recombinant DNA techniques (a transgeniccell), or which nucleic acid not present in said wild-type is the resultof one or more mutations—for example using recombinant DNA techniques oranother mutagenesis technique such as UV-irradiation—in a nucleic acidsequence present in said wild-type (such as a gene encoding a wild-typepolypeptide) or wherein the nucleic acid sequence of a gene has beenmodified to target the polypeptide product (encoding it) towards anothercellular compartment. Further, the term “recombinant (cell)” inparticular relates to a strain (cell) from which DNA sequences have beenremoved using recombinant DNA techniques.

The term “transgenic (yeast) cell” as used herein, refers to a strain(cell) containing nucleic acid not naturally occurring in that strain(cell) and which has been introduced into that strain (cell) usingrecombinant DNA techniques, i.e. a recombinant cell).

The term “mutated” as used herein regarding proteins or polypeptidesmeans that at least one amino acid in the wild-type or naturallyoccurring protein or polypeptide sequence has been replaced with adifferent amino acid, inserted or deleted from the sequence viamutagenesis of nucleic acids encoding these amino acids. Mutagenesis isa well-known method in the art, and includes, for example, site-directedmutagenesis by means of PCR or via oligonucleotide-mediated mutagenesisas described in Sambrook et al., Molecular Cloning-A Laboratory Manual,2nd ed., Vol. 1-3 (1989). The term “mutated” as used herein regardinggenes means that at least one nucleotide in the nucleic acid sequence ofthat gene or a regulatory sequence thereof, has been replaced with adifferent nucleotide, or has been deleted from the sequence viamutagenesis, resulting in the transcription of a protein sequence with aqualitatively of quantitatively altered function or the knock-out ofthat gene.

In the context of this invention an “altered gene” has the same meaningas a mutated gene.

The term “gene”, as used herein, refers to a nucleic acid sequencecontaining a template for a nucleic acid polymerase, in eukaryotes, RNApolymerase II. Genes are transcribed into mRNAs that are then translatedinto protein.

The term “nucleic acid” as used herein, includes reference to adeoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, ineither single or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e. g., peptidenucleic acids). A polynucleotide can be full-length or a subsequence ofa native or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulphation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation.

When an enzyme is mentioned with reference to an enzyme class (EC), theenzyme class is a class wherein the enzyme is classified or may beclassified, on the basis of the Enzyme Nomenclature provided by theNomenclature Committee of the International Union of Biochemistry andMolecular Biology (NC-IUBMB), which nomenclature may be found atchem.qmuLac.uk/iubmb/enzyme. Other suitable enzymes that have not (yet)been classified in a specified class but may be classified as such, aremeant to be included.

If referred herein to a protein or a nucleic acid sequence, such as agene, by reference to a accession number, this number in particular isused to refer to a protein or nucleic acid sequence (gene) having asequence as can be found via ncbi.nlm.nih.gov, (as available on 14 Jun.2016) unless specified otherwise.

Every nucleic acid sequence herein that encodes a polypeptide also, byreference to the genetic code, describes every possible silent variationof the nucleic acid. The term “conservatively modified variants” appliesto both amino acid and nucleic acid sequences. With respect toparticular nucleic acid sequences, conservatively modified variantsrefers to those nucleic acids which encode identical or conservativelymodified variants of the amino acid sequences due to the degeneracy ofthe genetic code. The term “degeneracy of the genetic code” refers tothe fact that a large number of functionally identical nucleic acidsencode any given protein. For instance, the codons GCA, GCC, GCG and GCUall encode the amino acid alanine. Thus, at every position where analanine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation.

The term “functional homologue” (or in short “homologue”) of apolypeptide having a specific sequence (e.g. “SEQ ID NO: X”), as usedherein, refers to a polypeptide comprising said specific sequence withthe proviso that one or more amino acids are substituted, deleted,added, and/or inserted, and which polypeptide has (qualitatively) thesame enzymatic functionality for substrate conversion. Thisfunctionality may be tested by use of an assay system comprising arecombinant cell comprising an expression vector for the expression ofthe homologue in yeast, said expression vector comprising a heterologousnucleic acid sequence operably linked to a promoter functional in theyeast and said heterologous nucleic acid sequence encoding thehomologous polypeptide of which enzymatic activity for convertingacetyl-Coenzyme A to acetaldehyde in the cell is to be tested, andassessing whether said conversion occurs in said cells. Candidatehomologues may be identified by using in silico similarity analyses. Adetailed example of such an analysis is described in Example 2 ofWO2009/013159. The skilled person will be able to derive there from howsuitable candidate homologues may be found and, optionally uponcodon(pair) optimization, will be able to test the requiredfunctionality of such candidate homologues using a suitable assay systemas described above. A suitable homologue represents a polypeptide havingan amino acid sequence similar to a specific polypeptide of more than50%, preferably of 60% or more, in particular of at least 70%, more inparticular of at least 80%, at least 90%, at least 95%, at least 97%, atleast 98% or at least 99% and having the required enzymaticfunctionality. With respect to nucleic acid sequences, the termfunctional homologue is meant to include nucleic acid sequences whichdiffer from another nucleic acid sequence due to the degeneracy of thegenetic code and encode the same polypeptide sequence.

Sequence identity is herein defined as a relationship between two ormore amino acid (polypeptide or protein) sequences or two or morenucleic acid (polynucleotide) sequences, as determined by comparing thesequences. Usually, sequence identities or similarities are comparedover the whole length of the sequences compared. In the art, “identity”also means the degree of sequence relatedness between amino acid ornucleic acid sequences, as the case may be, as determined by the matchbetween strings of such sequences.

Amino acid or nucleotide sequences are said to be homologous whenexhibiting a certain level of similarity. Two sequences being homologousindicate a common evolutionary origin. Whether two homologous sequencesare closely related or more distantly related is indicated by “percentidentity” or “percent similarity”, which is high or low respectively.Although disputed, to indicate “percent identity” or “percentsimilarity”, “level of homology” or “percent homology” are frequentlyused interchangeably. A comparison of sequences and determination ofpercent identity between two sequences can be accomplished using amathematical algorithm. The skilled person will be aware of the factthat several different computer programs are available to align twosequences and determine the homology between two sequences (Kruskal, J.B. (1983) An overview of sequence comparison In D. Sankoff and J. B.Kruskal, (ed.), Time warps, string edits and macromolecules: the theoryand practice of sequence comparison, pp. 1-44 Addison Wesley). Thepercent identity between two amino acid sequences can be determinedusing the Needleman and Wunsch algorithm for the alignment of twosequences. (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48,443-453). The algorithm aligns amino acid sequences as well asnucleotide sequences. The Needleman-Wunsch algorithm has beenimplemented in the computer program NEEDLE. For the purpose of thisinvention the NEEDLE program from the EMBOSS package was used (version2.8.0 or higher, EMBOSS: The European Molecular Biology Open SoftwareSuite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16,(6) pp 276-277, emboss.bioinformatics.nl). For protein sequences,EBLOSUM62 is used for the substitution matrix. For nucleotide sequences,EDNAFULL is used. Other matrices can be specified. The optionalparameters used for alignment of amino acid sequences are a gap-openpenalty of 10 and a gap extension penalty of 0.5. The skilled personwill appreciate that all these different parameters will yield slightlydifferent results but that the overall percentage identity of twosequences is not significantly altered when using different algorithms.

The homology or identity is the percentage of identical matches betweenthe two full sequences over the total aligned region including any gapsor extensions. The homology or identity between the two alignedsequences is calculated as follows: Number of corresponding positions inthe alignment showing an identical amino acid in both sequences dividedby the total length of the alignment including the gaps. The identitydefined as herein can be obtained from NEEDLE and is labelled in theoutput of the program as “IDENTITY”.

The homology or identity between the two aligned sequences is calculatedas follows: Number of corresponding positions in the alignment showingan identical amino acid in both sequences divided by the total length ofthe alignment after subtraction of the total number of gaps in thealignment. The identity defined as herein can be obtained from NEEDLE byusing the NOBRIEF option and is labeled in the output of the program as“longest-identity”.

A variant of a nucleotide or amino acid sequence disclosed herein mayalso be defined as a nucleotide or amino acid sequence having one orseveral substitutions, insertions and/or deletions as compared to thenucleotide or amino acid sequence specifically disclosed herein (e.g. inde the sequence listing).

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine. In an embodiment, conservative aminoacids substitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine. Substitutional variants of the amino acid sequencedisclosed herein are those in which at least one residue in thedisclosed sequences has been removed and a different residue inserted inits place. Preferably, the amino acid change is conservative. In anembodiment, conservative substitutions for each of the naturallyoccurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Glnor His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly toPro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys toArg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr;Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Val to Ile or Leu.

Nucleotide sequences of the invention may also be defined by theircapability to hybridise with parts of specific nucleotide sequencesdisclosed herein, respectively, under moderate, or preferably understringent hybridisation conditions. Stringent hybridisation conditionsare herein defined as conditions that allow a nucleic acid sequence ofat least about 25, preferably about 50 nucleotides, 75 or 100 and mostpreferably of about 200 or more nucleotides, to hybridise at atemperature of about 65° C. in a solution comprising about 1 M salt,preferably 6×SSC or any other solution having a comparable ionicstrength, and washing at 65° C. in a solution comprising about 0.1 Msalt, or less, preferably 0.2×SSC or any other solution having acomparable ionic strength. Preferably, the hybridisation is performedovernight, i.e. at least for 10 hours and preferably washing isperformed for at least one hour with at least two changes of the washingsolution. These conditions will usually allow the specific hybridisationof sequences having about 90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow anucleic acid sequences of at least 50 nucleotides, preferably of about200 or more nucleotides, to hybridise at a temperature of about 45° C.in a solution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

“Expression” refers to the transcription of a gene into structural RNA(rRNA, tRNA) or messenger RNA (mRNA) with subsequent translation into aprotein.

As used herein, “heterologous” in reference to a nucleic acid or proteinis a nucleic acid or protein that originates from a foreign species, or,if from the same species, is substantially modified from its native formin composition and/or genomic locus by deliberate human intervention.For example, a promoter operably linked to a heterologous structuralgene is from a species different from that from which the structuralgene was derived, or, if from the same species, one or both aresubstantially modified from their original form. A heterologous proteinmay originate from a foreign species or, if from the same species, issubstantially modified from its original form by deliberate humanintervention.

The term “heterologous expression” refers to the expression ofheterologous nucleic acids in a host cell. The expression ofheterologous proteins in eukaryotic host cell systems such as yeast arewell known to those of skill in the art. A polynucleotide comprising anucleic acid sequence of a gene encoding an enzyme with a specificactivity can be expressed in such a eukaryotic system. In someembodiments, transformed/transfected cells may be employed as expressionsystems for the expression of the enzymes. Expression of heterologousproteins in yeast is well known. Sherman, F., et al., Methods in YeastGenetics, Cold Spring Harbor Laboratory (1982) is a well-recognized workdescribing the various methods available to express proteins in yeast.Two widely utilized yeasts are Saccharomyces cerevisiae and Pichiapastoris. Vectors, strains, and protocols for expression inSaccharomyces and Pichia are known in the art and available fromcommercial suppliers (e.g., Invitrogen). Suitable vectors usually haveexpression control sequences, such as promoters, including3-phosphoglycerate kinase or alcohol oxidase, and an origin ofreplication, termination sequences and the like as desired.

As used herein “promoter” is a DNA sequence that directs thetranscription of a (structural) gene. Typically, a promoter is locatedin the 5′-region of a gene, proximal to the transcriptional start siteof a (structural) gene. Promoter sequences may be constitutive,inducible or repressible. In an embodiment there is no (external)inducer needed.

The term “vector” as used herein, includes reference to an autosomalexpression vector and to an integration vector used for integration intothe chromosome.

The term “expression vector” refers to a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestunder the control of (i.e. operably linked to) additional nucleic acidsegments that provide for its transcription. Such additional segmentsmay include promoter and terminator sequences, and may optionallyinclude one or more origins of replication, one or more selectablemarkers, an enhancer, a polyadenylation signal, and the like. Expressionvectors are generally derived from plasmid or viral DNA, or may containelements of both. In particular an expression vector comprises a nucleicacid sequence that comprises in the 5′ to 3′ direction and operablylinked: (a) a yeast-recognized transcription and translation initiationregion, (b) a coding sequence for a polypeptide of interest, and (c) ayeast-recognized transcription and translation termination region.“Plasmid” refers to autonomously replicating extrachromosomal DNA whichis not integrated into a microorganism's genome and is usually circularin nature.

An “integration vector” refers to a DNA molecule, linear or circular,that can be incorporated in a microorganism's genome and provides forstable inheritance of a gene encoding a polypeptide of interest. Theintegration vector generally comprises one or more segments comprising agene sequence encoding a polypeptide of interest under the control of(i.e. operably linked to) additional nucleic acid segments that providefor its transcription. Such additional segments may include promoter andterminator sequences, and one or more segments that drive theincorporation of the gene of interest into the genome of the targetcell, usually by the process of homologous recombination. Typically, theintegration vector will be one which can be transferred into the targetcell, but which has a replicon which is nonfunctional in that organism.Integration of the segment comprising the gene of interest may beselected if an appropriate marker is included within that segment.

By “host cell” is meant a cell which contains a vector and supports thereplication and/or expression of the vector.

“Transformation” and “transforming”, as used herein, refers to theinsertion of an exogenous polynucleotide into a host cell, irrespectiveof the method used for the insertion, for example, direct uptake,transduction, f-mating or electroporation. The exogenous polynucleotidemay be maintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host cell genome.

By “disruption” is meant (or includes) all nucleic acid modificationssuch as nucleotide deletions or substitutions, gene knock-outs, (other)which affect the translation or transcription of the correspondingpolypeptide and/or which affect the enzymatic (specific) activity, itssubstrate specificity, and/or or stability. Such modifications may betargeted on the coding sequence or on the promotor of the gene.

The term “encoding” has the same meaning as “coding for”. Thus, by wayof example, “one or more heterologous genes encoding a glyceroldehydrogenase” has the same meaning as “one or more heterologous genescoding for a glycerol dehydrogenase”. As far as genes encoding an enzymeare concerned, the phrase “one or more heterologous genes encoding a X”,wherein X denotes an enzyme, has the same meaning as “one or moreheterologous genes encoding an enzyme having X activity”. Thus, by wayof example, “one or more heterologous genes encoding a glyceroldehydrogenase” has the same meaning as “one or more heterologous genesencoding an enzyme having glycerol dehydrogenase activity”.

In one aspect the invention provides a recombinant cell, preferably ayeast cell comprising:

-   -   a) one or more heterologous genes encoding a glycerol        dehydrogenase,    -   b) one or more genes encoding a dihydroxyacetone kinase (E.C.        2.7.1.28 and/or E.C. 2.7.1.29);    -   c) one or more heterologous genes encoding a        ribulose-1,5-biphosphate carboxylase oxygenase (RuBisCO; EC        4.1.1.39); and    -   d) one or more heterologous genes encoding a phosphoribulokinase        (EC 2.7.1.19, PRK); and optionally    -   e) one or more heterologous genes encoding a glycerol        transporter.

In an embodiment the glycerol dehydrogenase is preferably a NAD⁺ linkedglycerol dehydrogenase (EC 1.1.1.6). Such enzyme may be from bacterialorigin or for instance from fungal origin. An example is gldA from E.coli.

Alternatively, the glycerol dehydrogenase may be a NADP⁺ linked glyceroldehydrogenase (EC 1.1.1.72).

When the cell is used for ethanol production, which typically takesplace under anaerobic conditions, a NAD⁺ linked glycerol dehydrogenaseis preferred.

In an embodiment the cell comprises one or more nucleic acid sequencesencoding a heterologous glycerol dehydrogenase represented by amino acidsequence SEQ ID NO:13 or a functional homologue thereof a havingsequence identity of at least 50%, preferably at least 60%, 70%, 75%,80%, 85%, 90% or 95%.

In an embodiment the dihydroxy acetone kinase is encoded by anendogenous gene, e.g. a DAK1 gene, which endogenous gene is preferablyplaced under control of a constitutive promoter.

In an embodiment the cell comprises one or more nucleic acid sequencesencoding a dihydroxy acetone kinase represented by amino acid sequenceaccording to SEQ ID NO: 14 or by a functional homologue thereof having asequence identity of at least 50%, preferably at least 60%, 70%, 75%,80%, 85%, 90% or 95%, which gene is preferably placed under control of aconstitutive promoter.

The dihydroxy acetone kinase may also have glyceraldehyde kinaseactivity.

WO2014/129898 discloses a yeast cell comprising one or more genes codingfor ribulose-1,5-biphosphate carboxylase oxygenase (EC 4.1.1.39,RuBisCO); one or more genes coding for phosphoribulokinase (EC 2.7.1.19,PRK). The inventors have found that by introducing one or more genesencoding an NAD⁺ linked glycerol dehydrogenase (EC 1.1.1.6 or EC1.1.1.72), and one or more genes encoding a dihydroxyacetone kinase(E.C. 2.7.1.28 or E.C. 2.7.1.29); the ethanol yield can be increased.Glycerol may still be produced, but is, at least partially converted toethanol.

One advantage of this invention is that glycerol production is reducedand/or the ethanol yield is increased. Without wanting to be bound bytheory, the inventors think that this may be the result of re-oxidationof NADH by using CO₂ as electron acceptor (through RuBisCO), rather thanproduce glycerol.

In an embodiment, the cell comprises a genetic modification thatincreases the specific activity of dihydroxyacetone kinase in the cell.A dihydroxyacetone kinase is herein understood as an enzyme thatcatalyzes the chemical reaction ((EC 2.7.1.29):ATP+glycerone↔H ADP+glycerone phosphate

Other names in common use include glycerone kinase, ATP:glyceronephosphotransferase and (phosphorylating) acetol kinase. It is understoodthat glycerone and dihydroxyacetone are the same molecule. Preferablysaid genetic modification causes overexpression of a dihydroxyacetonekinase, e.g. by overexpression of a nucleotide sequence encoding adihydroxyacetone kinase. The nucleotide sequence encoding thedihydroxyacetone kinase may be endogenous to the cell or may be adihydroxyacetone kinase that is heterologous to the cell. Nucleotidesequences that may be used for overexpression of dihydroxyacetone kinasein the cells of the invention are e.g. the dihydroxyacetone kinase genesfrom S. cerevisiae (DAK1) and (DAK2) as e.g. described by Molin et al.(2003, J. Biol. Chem. 278:1415-1423). In a preferred embodiment acodon-optimised (see above) nucleotide sequence encoding thedihydroxyacetone kinase is overexpressed, such as e.g. a codon optimisednucleotide sequence encoding the dihydroxyacetone kinase of SEQ ID NO:14.

A preferred nucleotide sequence for overexpression of a dihydroxyacetonekinase is a nucleotide sequence encoding a dihydroxyacetone kinasecomprises an amino acid sequence with at least 45, 50, 60, 65, 70, 75,80, 85, 90, 95, 98, 99% amino acid sequence identity with SEQ ID NO: 14(S. cerevisiae (DAK1) or having one or several substitutions, insertionsand/or deletions as compared to SEQ ID NO: 14.

Nucleotide sequences that may be used for overexpression of aheterologous dihydroxyacetone kinase in the cells of the invention aree.g. sequences encoding bacterial dihydroxyacetone kinases such as thedhaK gene from Citrobacter freundii e.g. described by Daniel et al.(1995, J. Bacteriol. 177:4392-4401).

For overexpression of the nucleotide sequence encoding thedihydroxyacetone kinase, the nucleotide sequence (to be overexpressed)is placed in an expression construct wherein it is operably linked tosuitable expression regulatory regions/sequences to ensureoverexpression of the dihydroxyacetone kinase enzyme upon transformationof the expression construct into the host cell of the invention (seeabove). Suitable promoters for (over)expression of the nucleotidesequence coding for the enzyme having dihydroxyacetone kinase activityinclude promoters that are preferably insensitive to catabolite(glucose) repression, that are active under anaerobic conditions and/orthat preferably do not require xylose or arabinose for induction.Examples of such promoters are given above. A dihydroxyacetone kinase tobe overexpressed is preferably overexpressed by at least a factor 1.1,1.2, 1.5, 2, 5, 10 or 20 as compared to a strain which is geneticallyidentical except for the genetic modification causing theoverexpression. Preferably, the dihydroxyacetone kinase is overexpressedunder anaerobic conditions by at least a factor 1.1, 1.2, 1.5, 2, 5, 10or 20 as compared to a strain which is genetically identical except forthe genetic modification causing the overexpression. It is to beunderstood that these levels of overexpression may apply to the steadystate level of the enzyme's activity (specific activity in the cell),the steady state level of the enzyme's protein as well as to the steadystate level of the transcript coding for the enzyme in the cell.Overexpression of the nucleotide sequence in the host cell produces aspecific dihydroxyacetone kinase activity of at least 0.002, 0.005,0.01, 0.02 or 0.05 U min-1 (mg protein)-1, determined in cell extractsof the transformed host cells at 30° C. as described e.g. in theExamples of WO2013/081456.

In an embodiment the cell comprises a heterologous gene encoding adihydroxyacetone kinase. Suitable dihydroxyacetone kinases are fromSaccharomyces kudriavzevii, Zygosaccharomyces bailii, Kluyveromyceslactis, Candida glabrata, Yarrowia lipolytica, Klebsiella pneumoniae,Enterobacter aerogenes, Escherichia coli, Yarrowia lipolytica,Schizosaccharomyces pombe, Botryotinia fuckeliana, and Exophialadermatitidis.

The cell optionally comprises one or more heterologous genes encoding aglycerol transporter. In this embodiment any glycerol that is externallyavailable in the medium (e.g. from the backset in corn mash) or secretedafter internal cellular synthesis may be transported into the cell andconverted to ethanol.

In an embodiment the cell comprises a deletion or disruption of one ormore endogenous nucleotide sequences encoding a glycerol exporter (e.gFPS1).

In a further embodiment, the cell naturally lacks enzymatic activityneeded for the NADH-dependent glycerol synthesis, for example yeastcells belonging to the species Brettanomyces intermedius.

In an embodiment the cell comprises a deletion or disruption of one ormore endogenous nucleotide sequences encoding a glycerol 3-phosphatephosphohydrolase and/or encoding a glycerol-3-phosphate dehydrogenase.Such a deletion or disruption may result in decrease or removal ofenzymatic activity. The deleted or disrupted glycerol-3-phosphatedehydrogenase preferably belongs to EC 1.1.5.3, such as GUT2, or to EC1.1.1.8, such as PDP1 and or PDP2.

In embodiment the cell is free of genes encoding NADH-dependentglycerol-3-phosphate dehydrogenase.

In another embodiment the cell comprises a deletion or disruption of oneor more endogenous nucleotide sequences encoding a glycerol kinase (EC2.7.1.30). An example of such an enzyme is Gut1p.

The cell may be free of enzymatic activity needed for the NADH-dependentglycerol synthesis or has a reduced enzymatic activity with respect tothe NADH-dependent biochemical pathway for glycerol synthesis from acarbohydrate compared to its corresponding wild-type cell.

A reduced enzymatic activity can be achieved by modifying one or moregenes encoding a NAD-dependent glycerol 3-phosphate dehydrogenaseactivity (GPD) or one or more genes encoding a glycerol phosphatephosphatase activity (GPP), such that the enzyme is expressedconsiderably less than in the wild-type or such that the gene encodes apolypeptide with reduced activity. Such modifications can be carried outusing commonly known biotechnological techniques, and may in particularinclude one or more knock-out mutations or site-directed mutagenesis ofpromoter regions or coding regions of the structural genes encoding GPDand/or GPP.

Alternatively, strains that are defective in glycerol production may beobtained by random mutagenesis followed by selection of strains withreduced or absent activity of GPD and/or GPP. Examples of genes in S.cerevisiae encoding GPD-activity are GPD1, GPD2, and GPP-activity areGPP1 and GPP2.

GPD and/or GPP may be entirely deleted, or at least a part is deletedwhich encodes a part of the enzyme that is essential for its activity.In particular, good results have been achieved with a S. cerevisiaecell, wherein the open reading frames of the GPD1 gene and of the GPD2gene have been inactivated. Inactivation of a structural gene (targetgene) can be accomplished by a person skilled in the art bysynthetically synthesizing or otherwise constructing a DNA fragmentconsisting of a selectable marker gene flanked by DNA sequences that areidentical to sequences that flank the region of the host cell's genomethat is to be deleted. In particular, good results have been obtainedwith the inactivation of the GPD1 and GPD2 genes in Saccharomycescerevisiae by integration of the marker genes kanMX and hphMX4.Subsequently this DNA fragment is transformed into a host cell.Transformed cells that express the dominant marker gene are checked forcorrect replacement of the region that was designed to be deleted, forexample by a diagnostic polymerase chain reaction or Southernhybridization.

In an embodiment the cell comprises one or more nucleic acid sequencesencoding a heterologous glycerol transporter represented by amino acidsequence SEQ ID NO:7 or a functional homologue thereof having a sequenceidentity of at least 50%, preferably at least 60%, 70%, 75%, 80%, 85%,90% or 95%.

In an embodiment the cell comprises one or more nucleic acid sequencesencoding a heterologous glycerol transporter represented by amino acidsequence SEQ ID NO:8 or a functional homologue thereof having a sequenceidentity of at least 50%, preferably at least 60%, 70%, 75%, 80%, 85%,90% or 95%.

In an embodiment the cell a yeast cell. The cell may be selected fromSaccharomycetaceae, in particular from the group of Saccharomyces, suchas Saccharomyces cerevisiae; Kluyveromyces, such as Kluyveromycesmarxianus; Pichia, such as Pichia stipitis or Pichia angusta;Zygosaccharomyces, such as Zygosaccharomyces bailii; and Brettanomyces,such as Brettanomyces intermedius, Issatchenkia, such as Issatchenkiaorientalis and Hansenula.

In another embodiment the cell is a prokaryotic cell, such as selectedfrom the list consisting of Clostridium, Zymomonas, Thermobacter,Escherichia, Lactobacillus, Geobacillus and Bacillus.

In an embodiment the cell comprises one or more genes, preferably aheterologous genes, coding for molecular chaperones, said chaperonespreferably originating from a prokaryote, more preferably a bacterium,even more preferably E. coli.

Chaperones—when expressed—are preferably capable of functionallyinteracting with an enzyme in the microorganism, in particular with atleast one of Rubisco and PRK. Chaperones are proteins that providefavourable conditions for the correct folding of other proteins, thuspreventing aggregation. Newly made proteins usually must fold from alinear chain of amino acids into a three-dimensional form. Chaperoninsbelong to a large class of molecules that assist protein folding, calledmolecular chaperones. The energy to fold proteins is supplied byadenosine triphosphate (ATP). A review article about chaperones that isuseful herein is written by Yebenes (2001); “Chaperonins: two rings forfolding”; Hugo Yebenes et al. Trends in Biochemical Sciences, August2011, Vol. 36, No. 8.

In an embodiment, the one or more chaperone is from a bacterium, morepreferably from Escherichia, in particular E. coli GroEL and GroES fromE. coli may in particular encoded in a microorganism according to theinvention. Other preferred chaperones are chaperones from Saccharomyces,in particular Saccharomyces cerevisiae Hsp10 and Hsp60. If thechaperones are naturally expressed in an organelle such as amitochondrion (examples are Hsp60 and Hsp10 of Saccharomyces cerevisiae)relocation to the cytosol can be achieved e.g. by modifying the nativesignal sequence of the chaperonins.

In eukaryotes the proteins Hsp60 and Hsp10 are structurally andfunctionally nearly identical to GroEL and GroES, respectively. Thus, itis contemplated that Hsp60 and Hsp10 from any eukaryotic cell may serveas a chaperone for the Rubisco. See Zeilstra-Ryalls J, Fayet O,Georgopoulos C (1991). “The universally conserved GroE (Hsp60)chaperonins”. Annu Rev Microbiol. 45: 301-25.doi:10.1146/annurev.mi.45.100191.001505. PMID 1683763 and Horwich A L,Fenton W A, Chapman E, Farr G W (2007). “Two Families of Chaperonin:Physiology and Mechanism”. Annu Rev Cell Dev Biol. 23: 115-45.doi:10.1146/annurev.cellbio.23.090506.123555. PMID 17489689.

As an alternative to GroEL a functional homologue of GroEL may bepresent, in particular a functional homologue comprising an amino acidsequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequenceidentity with SEQ ID NO: 10. Suitable natural chaperones polypeptideshomologous to SEQ ID NO: 10 are given in Table 4 of WO2014/129898.

As an alternative to GroES a functional homologue of GroES may bepresent, in particular a functional homologue comprising an amino acidsequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequenceidentity with SEQ ID NO: 9. Suitable natural chaperones polypeptideshomologous to SEQ ID NO: 9 are given in Table 3 of WO2014/129898.

In an embodiment, a 10 kDa chaperone from Table 3 of WO2014/129898 iscombined with a matching 60 kDa chaperone from Table 4 fromWO2014/129898 of the same organism genus or species for expression inthe host. For instance: >gi|189189366|ref|XP_001931022.1|:71-168 10 kDachaperonin [Pyrenophora tritici-repentis] expressed together withmatching >gi|189190432|ref|XP_001931555.1| heat shock protein 60,mitochondrial precursor [Pyrenophora tritici-repentis Pt-1C-BFP].

All other combinations from Table 3 and 4 of WO2014/129898 similarlymade with same organism source are also available to the skilled personfor expression.

The RuBisCO may in principle be selected from eukaryotic and prokaryoticRuBisCO's. The RuBisCO is preferably from a non-phototrophic organism.In particular, the RuBisCO may be from a chemolithoautotrophicmicroorganism. Good results have been achieved with a bacterial RuBisCO.Preferably, the bacterial RuBisCO originates from a Thiobacillus, inparticular, Thiobacillus denitrificans, which is chemolithoautotrophic.The RuBisCO may be a single-subunit RuBisCO or a RuBisCO having morethan one subunit. In particular, good results have been achieved with asingle-subunit RuBisCO.

In particular, good results have been achieved with a form-II RuBbisCO,more in particular CbbM.

SEQ ID NO: 11 shows a sequence of a RuBisCO. It is encoded by the cbbMgene from Thiobacillus denitrificans. An alternative to this Rubisco isa functional homologue of this RuBisCO, in particular such functionalhomologue comprising an amino acid sequence having at least 80%, 85%,90% or 95% sequence identity with SEQ ID NO: 11. Suitable naturalRuBisCO polypeptides are given in Table 1 of WO2014/129898.

The RuBisCO is preferably functionally expressed in the cell, at leastduring use in an industrial process for preparing a compound ofinterest.

In an embodiment the functionally expressed RuBisCO has an activity,defined by the rate of ribulose-1,5-bisphosphate-dependent¹⁴C-bicarbonate incorporation by cell extracts of at least 1nmol·min⁻¹·(mg protein)⁻¹, in particular an activity of at least 2nmol·min⁻¹·(mg protein)⁻¹, more in particular an activity of at least 4nmol·min⁻¹·(mg protein)⁻¹. The upper limit for the activity is notcritical. In practice, the activity may be about 200 nmol·min⁻¹·(mgprotein)⁻¹ or less, in particular 25 nmol·min⁻¹·(mg protein)⁻¹, more inparticular 15 nmol·min⁻¹·(mg protein)⁻¹ or less, e.g. about 10nmol·min⁻¹·(mg protein)⁻¹ or less. The conditions for an assay fordetermining this Rubisco activity are as found in Example 4 ofWO2014/129898.

In an embodiment the PRK is originating from a plant selected fromCaryophyllales, in particular from Amaranthaceae, in particular fromSpinacia.

In an embodiment the cell comprises one or more nucleic acid sequencesencoding a PRK represented by amino acid sequence represented by SEQ IDNO: 12 or by a functional homologue thereof having sequence identity ofat least 50%, preferably at least 60%, 70%, 75%, 80%, 85%, 90% or 95%.

A functionally expressed phosphoribulokinase (PRK, EC 2.7.1.19) iscapable of catalysing the chemical reaction:ATP+D-ribulose 5-phosphate ADP+D-ribulose 1,5-bisphosphate  (I)

Thus, the two substrates of this enzyme are ATP and D-ribulose5-phosphate, whereas its two products are ADP and D-ribulose1,5-bisphosphate.

PRK belongs to the family of transferases, specifically thosetransferring phosphorus-containing groups (phosphotransferases) with analcohol group as acceptor. The systematic name of this enzyme class isATP:D-ribulose-5-phosphate 1-phosphotransferase. Other names in commonuse include phosphopentokinase, ribulose-5-phosphate kinase,phosphopentokinase, phosphoribulokinase (phosphorylating),5-phosphoribulose kinase, ribulose phosphate kinase, PKK, PRuK, and PRK.This enzyme participates in carbon fixation.

The PRK can be from a prokaryote or a eukaryote. Good results have beenachieved with a PRK originating from a eukaryote. Preferably theeukaryotic PRK originates from a plant selected from Caryophyllales, inparticular from Amaranthaceae, more in particular from Spinacia.

As an alternative to PRK from Spinacia a functional homologue of PRKfrom Spinacia may be present, in particular a functional homologuecomprising a sequence having at least 70%, 75%, 80%. 85%, 90% or 95%sequence identity with the PRK from Spinacia.

The one or more PRK genes may be under the control of a promoter (the“PRK promoter”) that enables higher expression under anaerobicconditions than under aerobic conditions.

In an embodiment the PRK promoter is ROX1 repressed. ROX1 is hereinhaeme-dependent repressor of hypoxic gene(s); that mediates aerobictranscriptional repression of hypoxia induced genes such as COX5b andCYC7; the repressor function is regulated through decreased promoteroccupancy in response to oxidative stress; and contains an HMG domainthat is responsible for DNA bending activity; involved in thehyperosmotic stress resistance. ROX1 is regulated by oxygen.

According to Kwast et al. (in: Genomic Analysis of Anaerobically inducedgenes in Saccharomyces cerevisiae: Functional roles of ROX1 and otherfactors in mediating the anoxic response, 2002, Journal of bacteriologyvol 184, no 1 p 250-265): “Although Rox1 functions in an O₂-independentmanner, its expression is oxygen (haeme) dependent, activated by thehaeme-dependent transcription factor Hap1 [Keng, T 1992. HAP1 and ROX1form a regulatory pathway in the repression of HEM13 transcription inSaccharomyces cerevisiae. Mol. Cell. Biol. 12: 2616-2623]. Thus, asoxygen levels fall to those that limit haeme biosynthesis [Labbe-Bois,R., and P. Labbe. 1990. Tetrapyrrole and heme biosynthesis in the yeastSaccharomyces cerevisiae, p. 235-285. In H. A. Dailey (ed.),Biosynthesis of heme and chlorophylls. McGraw-Hill, New York, N. Y],ROX1 is no longer transcribed [Zitomer, R. S., and C. V. Lowry. 1992.Regulation of gene expression by oxygen in Saccharomyces cerevisiae.Microbiol. Rev. 56:1-11], its protein levels fall [Zitomer, R. S., P.Carrico, and J. Decked. 1997. Regulation of hypoxic gene expression inyeast. Kidney Int. 51:507-513], and the genes it regulates arede-repressed.”

In an embodiment, the PRK promoter is ROX1-repressed. In an embodiment,the PRK promoter has one or more ROX1 binding motif.

In an embodiment, the PRK promoter comprises in its sequence one or moreof the motif according to SEQ ID NO: 15.

In an embodiment, the PRK promoter is the native promoter of a geneselected from the list consisting of: FET4, ANB1, YHR048W, DAN1, AAC3,TIR2, DIP5, HEM13, YNR014W, YAR028W, FUN 57, COX5B, OYE2, SUR2, FRDS1,PIS1, LAC1, YGR035C, YAL028W, EUG1, HEM14, ISU2, ERG26, YMR252C andSML1, in particular FET4, ANB1, YHR048W, DAN1, AAC3, TIR2, DIP5 andHEM13.

In an embodiment, the PRK promoter comprises in its sequence one or moreof the motif: TCGTTYAG and/or according to SEQ ID NO: 16.

In particular such PRK promoter is native promoter of a DAN, TIR or PAUgene. In an embodiment, the PRK promoter is the native promoter of agene selected from the list consisting of: TIR2, DAN1, TIR4, TIR3, PAU7,PAU5, YLL064C, YGR294W, DAN3, YIL176C, YGL261C, YOL161C, PAU1, PAU6,DAN2, YDR542W, YIR041W, YKL224C, PAU3, YLL025W, YOR394W, YHL046C,YMR325W, YAL068C, YPL282C, PAU2, PAU4, in particular the PRK promoter isthe native promoter of a gene selected from the list consisting of:TIR2, DAN1, TIR4, TIR3, PAU7, PAU5, YLL064C, YGR294W, DAN3, YIL176C,YGL261C, YOL161C, PAU1, PAU6, DAN2, YDR542W, YIR041W, YKL224C, PAU3,YLL025W.

In an embodiment, the promoter has a PRK expression ratioanaerobic/aerobic of 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more or 50 ormore.

As used herein “promoter” is a DNA sequence that directs thetranscription of a (structural) gene, herein in particular one or morephosphoribulokinase gene. The promoter enables higher expression duringanaerobic conditions than under aerobic conditions.

In an embodiment, the PRK promoter may be a synthetic oligonucleotide.It may be a product of artificial oligonucleotide synthesis. Artificialoligonucleotide synthesis is a method in synthetic biology that is usedto create artificial oligonucleotides, such as genes, in the laboratory.Commercial gene synthesis services are now available from numerouscompanies worldwide, some of which have built their business modelaround this task. Current gene synthesis approaches are most often basedon a combination of organic chemistry and molecular biologicaltechniques and entire genes may be synthesized “de novo”, without theneed for precursor template DNA.

In an embodiment, the promoter is located in the 5′ region of a the PRKgene, In an embodiment it is located proximal to the transcriptionalstart site of PRK gene.

The invention further relates to a vector (as defined hereinafter)comprising PRK and a promoter that enables higher expression duringanaerobic conditions than under aerobic conditions.

The PRK promoter may have a PRK expression ratio anaerobic/aerobic of 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 20 or more or 50 or more.

In an embodiment the PRK promoter is a synthetic oligonucleotide. ThePRK promoter preferably enables expression only during anaerobicconditions.

A suitable PRK promotor is ANB1 and/or DAN1 as mentioned inEP16174382.8.

The cell may contain genes of a pentose metabolic pathway non-native tothe cell and/or that allow the recombinant cell to convert pentose(s).In one embodiment, the cell may comprise one or two or more copies ofone or more xylose isomerases and/or one or two or more copies of one ormore xylose reductase and xylitol dehydrogenase genes, allowing therecombinant cell to convert xylose. In an embodiment thereof, thesegenes may be integrated into the recombinant cell genome. In anotherembodiment, the recombinant cell comprises the genes araA, araB andaraD. It is then able to ferment arabinose. In one embodiment of theinvention the recombinant cell comprises xylA-gene, XYL1 gene and XYL2gene and/or XKS1-gene, to allow the recombinant cell to ferment xylose;deletion of the aldose reductase (GRE3) gene; overexpression of one ormore PPP-genes, e.g. TAL1, TAL2, TKL1, TKL2, RPE1 and RKI1 to allow theincrease of the flux through the pentose phosphate path-way in the cell,and/or overexpression of GAL2 and/or deletion of GALS®. Thus thoughinclusion of the above genes, suitable pentose or other metabolicpathway(s) may be introduced in the recombinant cell that werenon-native in the (wild type) recombinant cell.

In an embodiment, the following genes may be introduced in therecombinant cell by introduction into a host cell:

-   -   1) a set consisting of PPP-genes TAL1, TKL1, RPE1 and RKI1,        optionally under control of strong constitutive promoter;    -   2) a set consisting of a xylA-gene under control of strong        constitutive promoter;    -   3) a set comprising a XKS1-gene under control of strong        constitutive promoter,    -   4) a set consisting of the bacterial genes araA, araB and araD        under control of a strong constitutive promoter,    -   5) deletion of an aldose reductase gene

The above cells may be constructed using known recombinant expressiontechniques. The co-factor modification may be effected before,simultaneous or after any of the modifications 1-5 above.

The cell according to the invention may be subjected to evolutionaryengineering to improve its properties. Evolutionary engineeringprocesses are known processes. Evolutionary engineering is a processwherein industrially relevant phenotypes of a microorganism, herein therecombinant cell, can be coupled to the specific growth rate and/or theaffinity for a nutrient, by a process of rationally set-up naturalselection. Evolutionary Engineering is for instance described in detailin Kuijper, M, et al, FEMS, Eukaryotic cell Research 5(2005) 925-934,WO2008041840 and WO2009112472. After the evolutionary engineering theresulting pentose fermenting recombinant cell is isolated. The isolationmay be executed in any known manner, e.g. by separation of cells from arecombinant cell broth used in the evolutionary engineering, forinstance by taking a cell sample or by filtration or centrifugation.

In an embodiment, the cell is marker-free. As used herein, the term“marker” refers to a gene encoding a trait or a phenotype which permitsthe selection of, or the screening for, a host cell containing themarker. Marker-free means that markers are essentially absent in therecombinant cell. Being marker-free is particularly advantageous whenantibiotic markers have been used in construction of the recombinantcell and are removed thereafter. Removal of markers may be done usingany suitable prior art technique, e.g. intramolecular recombination.

In one embodiment, the cell is constructed on the basis of an inhibitortolerant host cell, wherein the construction is conducted as describedhereinafter. Inhibitor tolerant host cells may be selected by screeningstrains for growth on inhibitors containing materials, such asillustrated in Kadar et al, Appl. Biochem. Biotechnol. (2007), Vol.136-140, 847-858, wherein an inhibitor tolerant S. cerevisiae strainATCC 26602 was selected.

To increase the likelihood that enzyme activity is expressed atsufficient levels and in active form in the cell, the nucleotidesequence encoding these enzymes, as well as the RuBisCO enzyme and otherenzymes of the disclosure are preferably adapted to optimise their codonusage to that of the cell in question.

The adaptiveness of a nucleotide sequence encoding an enzyme to thecodon usage of a cell may be expressed as codon adaptation index (CAI).The codon adaptation index is herein defined as a measurement of therelative adaptiveness of the codon usage of a gene towards the codonusage of highly expressed genes in a particular cell or organism. Therelative adaptiveness (w) of each codon is the ratio of the usage ofeach codon, to that of the most abundant codon for the same amino acid.The CAI index is defined as the geometric mean of these relativeadaptiveness values. Non-synonymous codons and termination codons(dependent on genetic code) are excluded. CAI values range from 0 to 1,with higher values indicating a higher proportion of the most abundantcodons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295;also see: Jansen et al., 2003, Nucleic Acids Res. 31(8):2242-51). Anadapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8 or 0.9. Most preferred are the sequences whichhave been codon optimised for expression in the host cell in questionsuch as e.g. S. cerevisiae cells.

In an embodiment the invention provides a recombinant S. cerevisae cellcomprising:

-   -   a) one or more heterologous genes encoding a glycerol        dehydrogenase,    -   b) one or more genes encoding a dihydroxyacetone kinase (E.C.        2.7.1.28 and/or E.C. 2.7.1.29);    -   c) one or more heterologous genes encoding a        ribulose-1,5-biphosphate carboxylase oxygenase (RuBisCO; EC        4.1.1.39);    -   d) one or more heterologous genes encoding a phosphoribulokinase        (EC 2.7.1.19, PRK);    -   e) one or more heterologous genes encoding a molecular        chaperone; and    -   f) one or more heterologous genes encoding a glycerol        transporter;        wherein said cell comprises:    -   g) a deletion or disruption of one or more endogenous nucleotide        sequences encoding a glycerol-3-phosphate dehydrogenase.        In another embodiment the invention includes a recombinant S.        cerevisae cell comprising:    -   a) one or more heterologous genes encoding a glycerol        dehydrogenase represented by amino acid sequence SEQ ID NO: 13        or a functional homologue thereof a having sequence identity of        at least 50%;    -   b) one or more genes encoding a dihydroxyacetone kinase        represented by amino acid sequence according to SEQ ID NO: 14 or        by a functional homologue thereof having a sequence identity of        at least 50%, which gene is placed under control of a        constitutive promoter;    -   c) one or more heterologous genes encoding a RubisCo represented        by amino acid sequence according to SEQ ID NO: 11 or a        functional homologue thereof having a sequence identity of at        least 80%; and    -   d) one or more heterologous genes encoding groES represented by        amino acid sequence according to SEQ ID NO: 9 or a functional        homologue thereof having a sequence identity of at least 70%        and/or groEL represented by amino acid sequence according to SEQ        ID NO: 10 or a functional homologue thereof having a sequence        identity of at least 70%;    -   e) one or more heterologous genes encoding a PRK as represented        SEQ ID NO: 12 or a functional homologue thereof having a        sequence identity of at least 50%; and optionally    -   f) one or more genes heterologous encoding a glycerol        transporter represented by amino acid sequence according to SEQ        ID NO: 7 or by a functional homologue thereof having a sequence        identity of at least 50% and/or a glycerol transported        represented by amino acid sequence according to SEQ ID NO: 8 or        by a functional homologue thereof having a sequence identity of        at least 50%;        wherein said cell comprises:    -   g) a deletion or disruption of one or more endogenous nucleotide        sequences encoding a glycerol-3-phosphate dehydrogenase.

The invention further provides the use of a cell according to theinvention for preparation of ethanol.

The invention also provides the use of a cell according to the inventionfor preparation of succinic acid.

The invention further provides a process for preparing fermentationproduct, comprising preparing a fermentation product from a fermentablecarbohydrate, in particular selected from the group of glucose,fructose, sucrose, maltose, xylose, arabinose, galactose and mannosewhich preparation is carried out under anaerobic conditions using a cellaccording to the invention.

In an embodiment the fermentable carbohydrate is obtained from starch,lignocellulose, and/or pectin.

The starch, lignocellulose, and/or pectin may be contacted with anenzyme composition, wherein one or more sugar is produced, and whereinthe produced sugar is fermented to give a fermentation product, whereinthe fermentation is conducted with a cell of the invention.

The fermentation product may be one or more of ethanol, butanol,succinic acid, lactic acid, a plastic, an organic acid, a solvent, ananimal feed supplement, a pharmaceutical, a vitamin, an amino acid, anenzyme or a chemical feedstock.

The process is particularly useful when glycerol is fed externally tothe process, such as crude glycerol from transesterification-basedbiodiesel production or recirculation of backset, which is then taken upand converted to ethanol by the claimed cell.

EXAMPLES Material and Methods

General Molecular Biology Techniques

Unless indicated otherwise, the methods used are standard biochemicaltechniques. Examples of suitable general methodology textbooks includeSambrook et al., Molecular Cloning, a Laboratory Manual (1989) andAusubel et al., Current Protocols in Molecular Biology (1995), JohnWiley & Sons, Inc.

Plasmids, Oligonucleotide Primers and Strains

Plasmids used in the examples are listed in Table 1. Primers used in theexamples are listed in Table 2. Strains used for further strainengineering are listed in Table 3.

Media

Media used in the experiments were YEPh-medium (10 g/l yeast extract, 20g/l phytone) and solid YNB-medium (6.7 g/l yeast nitrogen base, 15 g/lagar), supplemented with sugars as indicated in the examples. For solidYEPh medium, 15 g/l agar was added to the liquid medium prior tosterilization.

In the microaerobic or anaerobic cultivation experiments, Mineral Mediumwas used. The composition of Mineral Medium has been described byVerduyn et al., (Yeast, 1992, volume 8, pp. 501-517). Ammoniumsulphatewas replaced by, 2.3 g/l urea as a nitrogen source. Initial pH of themedium was 4.6. In addition, for micro-/anaerobic experiments,ergosterol (0.01 g/L), Tween80 (0.42 g/L) and sugars (as indicated inexamples) were added.

Micro-/Anaerobic Cultivations

Strains were semi-aerobically propagated in a 100 mL Erlenmeyer shakeflask without baffle and with foam plug with 10 mL YEPh mediumsupplemented with 20 g/L glucose. Shake flasks were incubated 24 h at30° C. at a shaking speed of 280 rpm. Pre-cultured cells were pelleted,washed and re-suspended with 1 culture volume sterilized water. A volumeof re-suspended culture containing sufficient cell mass to inoculate themain fermentation medium to 75 mg of yeast (dry weight) per liter (seefurther below), was pelleted and re-suspended into main fermentationmedium.

To determine inoculum, a calibration curve was made of the IMX774 strainof biomass vs. OD600. This calibration curve was used to determine thevolume of re-suspended cell culture to be processed to inoculum for 75mg/L of yeast (dry weight).

Fermentation experiments were performed in an Alcoholic FermentationMonitor (AFM, Applikon, Delft, The Netherlands), using 500 ml bottlesfilled to 400 ml with Mineral Medium containing ca. 50 g/L glucose.Fermentation temperature was maintained at 32° C. and stirred at 250rpm, the pH was not controlled during fermentation. In addition to theonline recording of CO₂ production by the AFM (correlating with ethanol(EtOH)), samples were taken with at intervals of 4 hours during thefermentation to monitor yeast biomass, substrate utilization- andproduct formation. Total fermentation time was 32 hours.

Samples for HPLC analysis were separated from yeast biomass by passingthrough a 0.2 μm pore size filter.

HPLC Analysis

HPLC analysis was conducted as described in “Determination of sugars,byproducts and degradation products in liquid fraction in processsample”; Laboratory Analytical Procedure (LAP, Issue date: Dec. 8, 2006;by A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, and D.Templeton; Technical Report (NREL/TP-51042623); January 2008; NationalRenewable Energy Laboratory.

Strain Construction IMX774 and IME324

The strains generated and cultivated in the examples were transformantsof strain IMX581 or IMX774. IMX581 is a CEN.PK-based, Cas9-expressingstrain used for subsequent CRISPR-Cas9-mediated genome modifications(Mans et al., FEMS Yeast Res. 2015 March; 15(2). pii: fov004. doi:10.1093/femsyr/fov004). IMX774 is a CEN.PK-based strain expressing genesencoding the Calvin cycle enzymes phosphoribulokinase (S. oleacera prk)and the single subunit of ribulose-1,5-biphosphate-carboxylase (RuBisCO;Thiobacfflus denitrificans cbbM), and expressing genes encodingchaperonins (E. coli groEL and groES) to aid in the proper folding ofthe RuBisCO protein in the cytosol of S. cerevisiae. The strainconstruction of IMX774 has been described in European Patent applicationEP16174382.8 and description is also found below.

TABLE 1 Listing of plasmids used in examples Name Characteristics Originp426-TEF 2 μm ori, URA3, empty vector Mumberg D, et al., Gene, 1995 vol.156, pp. 119-122 pMEL10 2 μm ori, URA3, SNR52p-gRNA.CAN1- Mans et al.,FEMS Yeast Res. 2015 SUP4t March; 15(2). pii: fov004. pMEL11 2 μm ori,amdS, SNR52p-gRNA.CAN1- Mans et al., FEMS Yeast Res. 2015 SUP4t March;15(2). pii: fov004. pROS10 URA3, gRNA.CAN1-2 μm ori-gRNA.ADE2 Mans etal., FEMS Yeast Res. 2015 March; 15(2). pii: fov004. pUD232 Deliveryvector, TEF1p-groEL-ACT1t Guadalupe-Medina et al., Biotechnol Biofuels,2013, vol. 6, p. 125 pUD233 Delivery vector, TPI1p-groES-PGI1tGuadalupe-Medina et al., Biotechnol Biofuels, 2013, vol. 6, p. 125pUDE046 2 μm ori, GAL1p-prk-CYC1t Guadalupe-Medina et al., BiotechnolBiofuels, 2013, vol. 6, p. 125 pBTWW002 2 μm ori, URA3, TDH3p-cbbM-CYC1tGuadalupe-Medina et al., Biotechnol Biofuels, 2013, vol. 6, p. 125pUDR119 2 μm ori, amdS, SNR52p-gRNA.SGA1- EP16174382.8 SUP4t pUDR164 2μm ori, URA3, SNR52p-gRNA.X-2-SUP4t EP16174382.8 pUDR240 URA3,gRNA.GPD1-2 μm ori-gRNA.GPD2 EP16174382.8 pDB1332 Vector with cassetteENO1p-Ec_gldA-CYC1t Example 1; SEQ ID NO: 1 pDB1333 Vector with cassetteTPI1p-Sc_DAK1-ENO1t Example 1; SEQ ID NO: 2 pDB1334 Vector with cassetteADH1p-Dr_T3-TEF2t Example 1; SEQ ID NO: 3 pDB1336 Vector with cassettePRE3p-Zr_T5-TEF2t Example 1: SEQ ID NO 4 pRN1119 hphMX-bearing shuttlevector based on Example 2; SEQ ID NO: 5 pRS305

CRISPR/Cas9 genome editing was used to perform genetic modifications inIMX581 (resulting in IMX774) according to Mans et al. (2015; FEMS YeastRes. 2015 March; 15(2). pii: fov004. doi: 10.1093/femsyr/fov004). UniqueCRISPR/Cas9 sequences targeting SGA1 or X-2 were identified using apublicly available list (DiCarlo et al., Nucleic Acids Res. 2013; pp.1-8). For markerless genomic integration of gene cassettes, plasmidsexpressing unique gRNAs targeting the SGA1 locus or the intergenicregion X-2 (Mikkelsen et al., Metabolic Engineering, 2012, volume 14;pp. 101-111) were constructed. The plasmid backbones of pUDR119 andpURD164 were obtained by PCR amplification using the primer combination5792-5980 and plasmids pMEL11 and pMEL10, respectively, as templates.Phusion® Hot Start II High Fidelity DNA Polymerase (Thermo Scientific,Waltham, Mass., USA) was used for PCR amplification (e.g. forconstruction of plasmids and expression cassettes) in all cases,according to the manufacturer's guidelines. The plasmid inserts ofpUDR119 and pUDR164, containing the expression cassettes coding for theunique 20-bp gRNA sequences targeting SGA1 and X-2 respectively, wereobtained by PCR amplification using the primer combinations 5979-7023for SGA1 and 5979-7374 for X-2 and plasmids pMEL11 and pMEL10,respectively, as templates. The assembly of plasmids pUDR119 and pUDR164was performed in vitro using the Gibson Assembly® Cloning kit (NewEngland Biolabs, Ipswich, Mass., USA) following the supplier'sguidelines. The assembly was enabled by homologous sequences present atthe 5′ and 3′ ends of the PCR-amplified plasmid backbones and inserts.In each case, 1 μl of the Gibson-assembly mix was used for E. coli DH5atransformation by electroporation, performed in a Gene PulserXcellElectroporation System (Biorad, Hercules, Calif., USA). Correct assemblyof plasmids was confirmed by diagnostic PCR (Dreamtaq®, ThermoScientific) or restriction digestion. The constructed plasmids pUDR119and pUDR164 were isolated from transformed E. coli cultures using aSigma GenElute Plasmid kit (Sigma-Aldrich, St. Louis, Mo., USA) and usedfor transformation of S. cerevisiae.

A yeast expression cassette of cbbM was obtained by PCR amplificationusing plasmid pBTWW002 as template and primer combination 7549-7550. Theresulting fragment was ligated to a pJET/1.2 blunt vector(Thermo-Scientific) following the supplier's protocol and cloned to E.coli. The resulting plasmid was used as PCR template to generateintegration cbbM cassettes, using primer combinations 7074-7075,7548-6285, 6280-6273, 6281-6270, 6282-6271, 6284-6272, 6283-6275,6287-6276, 6288-6277, 6289-7075. The expression cassettes of cbbM weregenetically identical, except for different overhangs present at the 5′and 3′ ends of the fragments to allow for in vivo homologousrecombination. Yeast expression cassettes of groEL and groES wereobtained using plasmids pUD232 and pUD233 as templates and primercombinations 7076-7077 and 7078-7079, respectively. The genomicsequences corresponding to the DAN1 promoter (Knijnenburg et al., BMCGenomics. 2009; volume 10, p. 53), were obtained by PCR amplificationwith primer combinations 7930-7931 using genomic DNA of IMX585 astemplate. The terminator of PGK1 was obtained by PCR amplification withgenomic DNA of IMX585 as template using primer combinations 7084-7085and 7084-7934. The ORF of prk was obtained by PCR amplification usingprimer combinations 7932-7081 and plasmid pUDE046 as template. Theprimer combination resulted in prk-ORF fragments with homologousoverhangs to the DAN1 promoter sequence and the terminator of PGK1. Thecomplete expression cassette (DAN1p-prk-PGK1t) was assembled by in vivohomologous recombination after transformation to yeast and correctassembly was verified by diagnostic PCR. A complete list of all primersused in the examples is given in Table 2.

TABLE 2 Listing of oligonucleotide primers used in the examples withsequences Primer code SEQ ID Comment 5792 SEQ ID NO: 17 pUDR119 andpUDR164 construction 5980 SEQ ID NO: 18 pUDR119 and pUDR164 construction5979 SEQ ID NO: 19 pUDR119 and pUDR164 construction 7023 SEQ ID NO: 20pUDR119 construction 7374 SEQ ID NO: 21 pUDR164 construction 7549 SEQ IDNO: 22 Addition of 20 bp primer-binding sequence to cbbM 7550 SEQ ID NO:23 Addition of 20 bp primer-binding sequence to cbbM 7074 SEQ ID NO: 24cbbM cassette construction - D tag addition (single copycbbm-prk-chaperone integration) 7075 SEQ ID NO: 25 cbbM cassetteconstruction - J tag addition (single copy cbbm-prk-chaperoneintegration) 7548 SEQ ID NO: 26 cbbM cassette construction - SGA1 tagaddition 6285 SEQ ID NO: 27 cbbM cassette construction - G tag addition6280 SEQ ID NO: 28 cbbM cassette construction - A tag addition 6273 SEQID NO: 29 cbbM cassette construction - G tag addition 6281 SEQ ID NO: 30cbbM cassette construction - B tag addition 6270 SEQ ID NO: 31 cbbMcassette construction - A tag addition 6282 SEQ ID NO: 32 cbbM cassetteconstruction - C tag addition 6271 SEQ ID NO: 33 cbbM cassetteconstruction - B tag addition 6284 SEQ ID NO: 34 cbbM cassetteconstruction - D tag addition 6272 SEQ ID NO: 35 cbbM cassetteconstruction - C tag addition 6283 SEQ ID NO: 36 cbbM cassetteconstruction - D tag addition 6275 SEQ ID NO: 37 cbbM cassetteconstruction - M tag addition 6287 SEQ ID NO: 38 cbbM cassetteconstruction - M tag addition 6276 SEQ ID NO: 39 cbbM cassetteconstruction - N tag addition 6288 SEQ ID NO: 40 cbbM cassetteconstruction - N tag addition 6277 SEQ ID NO: 41 cbbM cassetteconstruction - O tag addition 6289 SEQ ID NO: 42 cbbM cassetteconstruction - O tag addition 7076 SEQ ID NO: 43 groEL cassetteconstruction - J tag addition 7077 SEQ ID NO: 44 groEL cassetteconstruction - H tag addition 7078 SEQ ID NO: 45 groES cassetteconstruction - H tag addition 7079 SEQ ID NO: 46 groES cassetteconstruction - SGA1 tag addition 7930 SEQ ID NO: 47 DAN1p prk cassetteconstruction 7931 SEQ ID NO: 48 DAN1p prk cassette construction 7084 SEQID NO: 49 prk cassette construction (PGK1t) 7085 SEQ ID NO: 50 prkcassette construction (PGK1t) - D tag addition (single copycbbm-prk-chaperone integration) 7934 SEQ ID NO: 51 prk cassetteconstruction (PGK1t) - X-2 tag addition 7081 SEQ ID NO: 52 prkamplification 7932 SEQ ID NO: 53 prk amplification (DAN1p cassette)BoZ-783 SEQ ID NO: 54 5′-INT1 amplification BoZ-788 SEQ ID NO: 553′-INT1 amplification DBC-13773 SEQ ID NO: 56 gRNA-INT1 amplificationDBC-13774 SEQ ID NO: 57 gRNA-INT1 amplification DBC-13775 SEQ ID NO: 58BB-1119 amplification DBC-13776 SEQ ID NO: 59 BB-1119 amplificationDBC-14041 SEQ ID NO: 60 DAK1 amplification DBC-14042 SEQ ID NO: 61 DAK1amplification DBC-14043 SEQ ID NO: 62 gldA amplication DBC-14044 SEQ IDNO: 63 gldA amplication DBC-14045 SEQ ID NO: 64 T3 amplificationDBC-14046 SEQ ID NO: 65 T5 amplification DBC-14048 SEQ ID NO: 66 T3amplification DBC-18463 SEQ ID NO: 67 5′-INT1 amplification DBC-18464SEQ ID NO: 68 3′-INT1 amplification

The lithium-acetate transformation protocol was used for yeasttransformations (Gietz & Woods, Methods Enzymol., 2002, pp. 87-96).Transformation mixtures were plated on Mineral Medium agar plates(Verduyn et al., Yeast, 1992, volume 8, pp. 501-517) (2% Bacto Agar, BD,Franklin Lakes, N.J., USA), supplemented with 20 g L⁻¹ glucose in thecase of transformations performed with pUDR164. In transformationsperformed with plasmid pUDR119, the agar plates were prepared asdescribed previously (Solis-Escalante, FEMS Yeast Res., 2013, volume 13,pp. 126-139). For the construction of strain IMX765 uracil wasadditionally supplemented to the agar plates (150 mg L⁻¹)(Sigma-Aldrich). Confirmation of the desired genotypes in each case wasperformed by diagnostic colony PCR. Recycling of pUDR164 was performedusing 5-fluoro-orotic acid (Zymo Research, Irvine, Calif., USA)counter-selection, following the supplier's guidelines. Recycling ofpUDR119 was performed as described previously (Solis-Escalante, FEMSYeast Res., 2013, volume 13, pp. 126-139). Strain IMX765 was obtained byco-transformation of pUDR119, the 9 abovementioned expression cassettesof cbbM with different connecting overhangs and the expression cassettesof groEL and groES to IMX581 (after plasmid recycling from the correctmutant). Overhangs present at the 5′ and 3′ ends of the moleculesallowed for in vivo assembly of the entire construct (11 fragments) andintegration in the SGA1 locus. Strain IMX774 was obtained bytransformation of strain IMX765 with the gRNA-expressing, X-2 targetingplasmid pUDR164 and the DAN1p, prk ORF, PGK1t fragments which wereassembled in vivo into the complete construct and subsequentlyintegrated in the X-2 locus. The control strain IME324 was obtained bytransformation of IMX581 with the empty vector p426-TEF. The genotypesof the strains is indicated in Table 3.

TABLE 3 Listing of S. cerevisiae strains used and generated in theexamples. Strain name Relevant Genotype Origin IME324 MATa ura3-52can1::cas9-natNT2 + p426-TEF. EP16174382.8 IMX581 MATa ura3-52can1::cas9-natNT2 Mans et al., FEMS Yeast Res. 2015 March; 15(2). pii:fov004. IMX585 MATa can1D::cas9-natNT2 URA3 TRP1 LEU2 HIS3 Mans et al.,FEMS Yeast Res. 2015 March; 5(2). pii: fov004. IMX765 MATa ura3-52can1::cas9-natNT2 sga1:: cbbM (9 copies), EP16174382.8 groES, groELIMX774 MATa ura3-52 can1::cas9-natNT2 sga1:: cbbM (9 copies),EP16174382.8 groES, groEL X-2::DAN1p-prk pUDR164 DS78742 MATa ura3-52can1::cas9-natNT2 sga1:: cbbM (9 copies), Example 2 groES, groELX-2::DAN1p-prk pUDR164 int1::TPI1p-DAK1-ENO1t, ENO1p-Ec_gldA-CYC1t,PRE3p- Zr_T5-TEF2t DS78743 MATa ura3-52 can1::cas9-natNT2 sga1:: cbbM (9copies), Example 2 groES, groEL X-2::DAN1p-prk pUDR164int1::TPI1p-DAK1-ENO1t, ENO1p-Ec_gldA-CYC1t, PRE3p- Zr_T5-TEF2t DS78744MATa ura3-52 can1::cas9-natNT2 sga1:: cbbM (9 copies), Example 2 groES,groEL X-2::DAN1p-prk pUDR164 int1::TPI1p-DAK1-ENO1t,ENO1p-Ec_gldA-CYC1t, ADH1p- Dr_T3-TEF2t

Example 1: Glycerol Reuptake Expression Cassette Construction

Expression Cassette Construction

The open reading frames (ORFs), promoter sequences and terminators weresynthesized at DNA 2.0 (Menlo Park, Calif. 94025, USA). The promoter,ORF and terminator sequences were recombined by using the Golden Gatetechnology, as described by Engler et al (2011) and references therein.The expression cassettes were cloned into a standard subcloning vector.The plasmids (listed in Table 1) containing the expression cassettesencoding the components of the glycerol re-uptake pathway are:

-   -   pDB1332 (SEQ ID NO: 1) bearing expression cassette for glycerol        dehydrogenase (EC 1.1.1.6) E. coli gldA under control of S.        cerevisiae ENO1 promoter and S. cerevisiae CYC1 terminator,    -   pDB1333 (SEQ ID NO: 2) bearing expression cassette for        dihydroxyacetone kinase (EC 2.7.1.29, EC 2.7.1.28) S. cerevisiae        DAK1 under control of S. cerevisiae TP/1 promoter and S.        cerevisiae ENO1 terminator,    -   pDB1334 (SEQ ID NO: 3) bearing expression cassette for glycerol        transporter/aquaporin D. rerio aqp9 (NP_001171215, hereforth        referenced as Dr_T3 or T3) under control of S. cerevisiae ADH1        promoter and S. cerevisiae TEF2 terminator,    -   pDB1336 (SEQ ID NO: 4) bearing expression cassette for glycerol        transporter Z. rouxii ZYRO0E01210p (hereforth referenced as        Zr_T5 or T5) under control of S. cerevisiae PRE3 promoter and S.        cerevisiae TEF2 terminator.

Example 2: Strain Construction DS78742, DS78743 and DS78744

Approach

The followed strain construction approach is described in patentapplication PCT/EP2013/056623 and PCT/EP2016/050136. PCT/EP2013/056623describes the techniques enabling the construction of expressioncassettes from various genes of interest in such a way, that thesecassettes are combined into a pathway and integrated in a specific locusof the yeast genome upon transformation of this yeast. PCT/EP2016/050136describes the use of a CRISPR-Cas9 system for integration of expressioncassettes into the genome of a host cell, in this case S. cerevisiae. Inthe construction of IMX774 a S. pyogenes Cas9 expression cassette wasalready integrated at the CAN1 locus. Upon introduction of an in vivoassembled gRNA-expressing plasmid and repair DNA fragments the intendedmodifications were made. Firstly, an integration site in the yeastgenome was selected. DNA fragments of approximately 500 bp of the up-and downstream parts of the integration locus were amplified by PCRusing primers introducing connectors to the generated PCR products.These connectors (50 bp in size) allow for correct in vivo recombinationof the pathway upon transformation in yeast. Secondly, the genes ofinterest, are amplified by PCR, incorporating a different connector(compatible with the connector on the of the neighbouring biobrick) ateach flank. Upon transformation of yeast cells with the DNA fragments,in vivo recombination and integration into the genome takes place at thedesired location. This technique facilitates parallel testing ofmultiple genetic designs, as one or more genes from the pathway can bereplaced with (an) other gene(s) or genetic element(s), as long as thatthe connectors that allow for homologous recombination remain constantand compatible with the preceeding and following biobrick in the design(patent application PCT/EP2013/056623).

Integration site: the expression cassettes were targeted at the INT1locus. The INT1 integration site is a non-coding region between NTR1(YOR071c) and GYP1 (YOR070c) located on chromosome XV of S cerevisiae.The guide sequence to target INT1 was designed with a gRNA designer tool(www.dna20.com/eCommerce/cas9/input).

The gRNA expression cassette (as described by DiCarlo et al., NucleicAcids Res. 2013; pp. 1-8) was ordered as synthetic DNA cassette (gBLOCK)at Integrated DNA Technologies (Leuven, Belgium) (INT1 gBLOCK; SEQ IDNO: 6). In vivo assembly of the gRNA expression plasmid is thencompleted by co-transforming a linear fragment derived from yeast vectorpRN1119. pRN11119 is a multi-copy yeast shuttling vector that contains afunctional hphMX marker cassette conferring resistance againstHygromycin B (HygB). The backbone of this plasmid is based on pRS305(Sikorski and Hieter, Genetics 1989, vol. 122, pp. 19-27), including afunctional 2 micron ORI sequence and a functional hphMX marker cassette(SEQ ID NO: 5, Table 1).

Transformation of IMX774 with Specified DNA Fragments Upon AssemblyComprising Glycerol Reuptake Pathway Designs

Strain IMX774 was transformed with the following fragments resulting theassembly of the glycerol reuptake pathway as depicted in FIG. 1:

-   -   1) a PCR fragment (5′-INT1) generated with primers BoZ-783 and        DBC-18463 with genomic DNA of strain CEN.PK113-7D as template;    -   2) a PCR fragment (DAK1) generated with primers DBC-14041 and        DBC-14042 using pDB1333 (SEQ ID NO: 1) as template;    -   3) a PCR fragment (gldA) generated with primers DBC-14043 and        DBC-DBC-14044 using pDB1332 (SEQ ID NO: 2) as template;    -   4) a PCR fragment (T3) generated with primers DBC-14045 and        DBC-14048 using pDB1334 (SEQ ID NO: 3) as template; or a PCR        fragment (T5) generated with primers DBC-14046 and DBC-14048        using pDB1336 (SEQ ID NO: 4) as template;    -   5) a PCR fragment (3′-INT1) generated with primers DBC-18464 and        BoZ-788 using genomic DNA of strain CEN.PK113-7D as template;    -   6) a PCR fragment (BB-1119) generated with primers DBC-13775 and        DBC-13776 using pRN1119 (SEQ ID NO: 5) as template;    -   7) a PCR fragment (gRNA-INT1) generated with primers DBC-13773        and DBC-13774 using INT1 gRNA (SEQ ID NO: 6) as template;

Transformants were selected on YEPh-agar plates containing 20 g/Lglucose and 200 μg HygB/ml. Diagnostic PCR was performed to confirm thecorrect assembly and integration at the INT1 locus of the pathway withT5 in strains DS78742 and DS78743 and with T3 in DS78744 (see Table 3for genotypes).

Example 3: Fermentation Experiment

Propagation of Strains

Strains IME324, IMX774, DS78742, DS784743 and DS78744 were pre-grown at30° C. and 280 rpm overnight under semi-aerobic conditions in MineralMedium supplemented with 20 g/L glucose supplemented with 0.05 g/Luracil.

Preparation of AFM Experiment

The following day, the optical density at 600 nm was determined andcells were spun down by centrifugation. Four hundred ml of MineralMedium containing approximately 50 grams of glucose per liter and 0.05g/L uracil was inoculated with one the abovementioned strains to 0.075g/L (dry weight). At specific time intervals samples were taken in orderto measure biomass, residual sugars, glycerol and acetic acid, as wellas the formation of ethanol.

Results Fermentation Experiment

The glycerol yield on glucose of strains IMX774, DS78742, DS78743 andDS78744 were 0.036, 0.014, 0.015, and 0.021 g/g, respectively, whichcorresponds to a 35%, 75%, 73% and 62%, respectively, decrease comparedto the reference strain IME324 (Table 4, FIG. 2). A decrease of glycerolproduction can be expected when NAD⁺ is, at least partly, regeneratedvia the RuBisCO pathway. The glycerol that is produced by the strain,since the Gpd1/Gpd2/Gpp1/Gpp2 pathway is left intact, and possiblyalready secreted by the cell is taken up again by either the T5 or T3glycerol transporters and re-shuttled to glycolysis, and, subsequently,ethanol fermentation, by the concerted action of gldA and DAK1. There-shuttling of glycerol to ethanol comes at the cost of 1 ATP andyields one NADH per mole of glycerol that is available for re-oxidationvia the Prk-RuBisCO pathway, thereby increasing the flux through thispathway further, and effectively decreasing the net glycerol produced infermentation. As a combined result of the decrease in glycerolproduction, CO₂ fixation via the Prk-RuBisCO pathway, and a decrease inbiomass yield, the engineered, RuBisCO expressing strain IMX774 produced3% more ethanol compared to the reference strain. Even more, theadditional re-shuttling of formed glycerol through the glycerol-reuptakepathway (T5/T3-gldA-DAK1) (Table 4, FIG. 2) by strains DS78742, DS78743and DS78744 resulted in a further increase towards ca. 6%, 7% and 5%,respectively, in ethanol yield compared to the reference strain on ca.50 g/L glucose in the experiments performed in this example.

TABLE 4 Fermentation yields and growth characteristics of strainsIME324, IMX774, DS78742, DS78743 and DS78744 on Mineral Mediumsupplemented with ca. 50 g/L glucose. Strain IME324 IMX774 DS78742DS78743 DS78744 Relevant genotype reference 9*cbbM, 9*cbbM, 9*cbbM,9*cbbM, DAN1p-prk, DAN1p-prk, DAN1p-prk, DAN1p-prk, groES, groEL groES,groEL, groES, groEL, groES, groEL, gldA, DAK1, T5 gldA, DAK1, T5 gldA,DAK1, T3 Y glycerol/glucose (g/g) 0.056 0.036 0.014 0.015 0.021 Ybiomass/glucose (g/g⁻¹) 0.085 0.048 0.040 0.045 0.042 Y EtOH/glucose(g/g) 0.387 0.398 0.409 0.414 0.408 Ratio glycerol 7.2 8.3 3.9 3.5 5.5produced/ biomass (mmol/g_(x))

The invention claimed is:
 1. A recombinant cell, optionally arecombinant yeast cell comprising: a) one or more heterologous genesencoding a glycerol dehydrogenase; b) one or more genes encoding adihydroxyacetone kinase (E.C. 2.7.1.28 and/or E.C. 2.7.1.29); c) one ormore heterologous genes encoding a ribulose-1,5-biphosphate carboxylaseoxygenase (EC 4.1.1.39, RuBisCO); and d) one or more heterologous genesencoding a phosphoribulokinase (EC 2.7.1.19, PRK); and optionally e) oneor more heterologous genes encoding a glycerol transporter.
 2. The cellaccording to claim 1 which comprises a deletion or disruption of one ormore endogenous nucleotide sequences encoding a glycerol exporter. 3.The cell according to claim 1 wherein the glycerol dehydrogenase is aNAD+linked glycerol dehydrogenase (EC 1.1.1.6) or a NADP⁺ linkedglycerol dehydrogenase (EC 1.1.1.72).
 4. The cell according to claim 1which comprises a genetic modification that increases the specificactivity of dihydroxyacetone kinase in the cell as compared to a cellwhich is genetically identical except for the modification causing theincreased specific activity of dihydroxyacetone kinase.
 5. The cellaccording to claim 1 which comprises a deletion or disruption of one ormore endogenous nucleotide sequences encoding a glycerol kinase (EC2.7.1.30).
 6. The cell according to claim 1 which comprises a deletionor disruption of one or more endogenous nucleotide sequences encoding aglycerol-3-phosphate dehydrogenase, which glycerol-3-phosphatedehydrogenase optionally belongs to EC 1.1.5.3, optionally gut2, or toEC 1.1.1.8, optionally PDP1/2, which cell is optionally free of genesencoding NADH-dependent glycerol 3-phosphate dehydrogenase.
 7. The cellaccording to claim 1 which comprises a deletion or disruption of one ormore endogenous nucleotide sequences encoding a glycerol 3-phosphatephosphohydrolase (GPP 1/2).
 8. The cell according to claim 1 whichcomprises one or more nucleic acid sequences encoding a heterologousglycerol transporter represented by amino acid sequence SEQ ID NO: 7 ora functional homologue thereof having sequence identity of at least 50%.9. The cell according to claim 1 which comprises one or more nucleicacid sequences encoding a heterologous glycerol transporter representedby amino acid sequence SEQ ID NO: 8 or a functional homologue thereofhaving sequence identity of at least 50%.
 10. The cell according toclaim 1 which is a yeast cell.
 11. The cell according to claim 1 whichis selected from Saccharomycetaceae, optionally from the group ofSaccharomyces, optionally Saccharomyces cerevisiae, Kluyveromyces,optionally Kluyveromyces marxianus, Pichia, optionally Pichia stipitisor Pichia angusta; Zygosaccharomyces optionally Zygosaccharomyces bailiiand Brettanomyces, optionally Brettanomyces intermedius, Issatchenkia,optionally Issatchenkia orientalis and Hansenula.
 12. The cell accordingto claim 1 further comprising one or more genes, optionally aheterologous genes, encoding molecular chaperones, said chaperonesoptionally originating from a prokaryote, optionally a bacterium,optionally E. coli, optionally said chaperones are selected from thegroup consisting of GroEL, GroES, functional homologues of GroEL, andfunctional homologues of GroES.
 13. The cell according to claim 1wherein the PRK is under control of a promoter (a PRK promoter) thatenables higher expression under anaerobic conditions than under aerobicconditions, which a PRK expression ratio anaerobic/aerobic of 2 or more,3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 ormore, 10 or more, 20 or more or 50 or more.
 14. A process for preparinga fermentation product, comprising preparing a fermentation product froma fermentable carbohydrate, optionally selected from the group ofglucose, fructose, sucrose, maltose, xylose, arabinose, galactose andmannose, wherein said preparing is carried out by contacting the cellaccording to claim 1 with the fermentable carbohydrate under anaerobicconditions.
 15. The process according to claim 14 wherein thefermentable carbohydrate is obtained from starch, lignocellulose, and/orpectin.
 16. The process according to claim 15, wherein the starch,lignocellulose, and/or pectin is contacted with an enzyme composition,wherein one or more sugar is produced, and wherein the produced sugar isfermented to give a fermentation product, wherein the fermentation isconducted with said cell.
 17. The process according to claim 15, whereinthe fermentation product is one or more of ethanol, butanol, lacticacid, succinic acid, a plastic, an organic acid, a solvent, an animalfeed supplement, a pharmaceutical, a vitamin, an amino acid, an enzymeor a chemical feedstock.